Spatial proteomics and cell-cell crosstalk analysis reveal PD-L1 and IL-6 interaction in human primary sclerosing cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is a chronic, immune-mediated cholangiopathy characterized by persistent inflammation and fibrotic remodelling of the biliary tree. We combined spatial proteomics of PSC-affected large bile ducts with a co-culture system of biliary epithelial cells (BECs) and activated peripheral blood mononuclear cells (aPBMCs) to dissect the molecular crosstalk at the epithelial-immune interface.

METHODS

Spatial proteomic analysis was performed on six PSC-affected bile ducts and five healthy controls by GeoMx® DSP. Single-cell RNA-sequencing analysis from publicly available datasets of human control (n = 22) and PSC-affected (n = 8) livers was performed. We developed an in vitro inflammatory model of PSC by transwell co-culture of BECs (i.e. H69 and primary human biliary tree stem cells) and primary human PBMCs (n = 6). Co-cultures were analysed by transcriptomics on nCounter®, by immunoassays and by culture with/without blocking agents.

RESULTS

Peribiliary glands showed higher PD-L1 expression in PSC (score = 1.2 ± 0.4) compared to controls (0.3 ± 0.5; p <0.05), which was associated with apoptosis in neighbouring immune cells on spatial proteomics. In vitro, aPBMCs induced the upregulation of 68 immunoregulatory genes in BECs; PD-L1 and IL-6 upregulation was confirmed at the protein level. BECs induced increased apoptosis in T (CD4+/CD8+) and NK cells. In co-culture, the exposure of BECs to the IL-6/sIL-6R complex induced significant PD-L1 upregulation (12.7 ± 1.3; p <0.001), which was inhibited by IL-6 receptor blockade (2.6 ± 1.2; p <0.001) or JAK1/2 inhibition.

CONCLUSIONS

These findings define a critical immunosuppressive axis in PSC driven by BEC-derived IL-6 and PD-L1. Mechanistically, IL-6 acts as an upstream regulator of PD-L1 via JAK/STAT3 and NF-κB signalling, predominantly through IL-6 trans-signalling. This study provides a rationale for targeting epithelial-immune cell interactions as a therapeutic strategy in PSC.

IMPACT AND IMPLICATIONS: The pathogenic basis of primary sclerosing cholangitis (PSC) is poorly understood, hampering the development of specific therapeutic approaches. We identified IL-6 as a key upstream regulator of PD-L1 via the JAK/STAT3 and NF-κB pathways in biliary epithelial cells, highlighting its central role in regulating epithelial-immune crosstalk during biliary injury. The identification of the PD-L1 pathway in epithelial-immune cell interactions in PSC may improve understanding of the disease, as well as inform the development of therapeutic strategies.